Methods and materials for detecting colorectal neoplasm

ABSTRACT

The present invention provides methods and materials related to the detection of colorectal neoplasm-specific markers in or associated with a subject&#39;s stool sample. In particular, the present invention provides methods and materials for identifying mammals having a colorectal neoplasm by detecting the presence of exfoliated epithelial markers (e.g., human DNA, tumor assoicated gene alterations, tumor associated proteins) and blood markers (e.g., homoglobin, serum proteins) in a stool sample obtained from the mammal.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 14/954,496, filed Nov. 30, 2015, which is a continuation ofabandoned U.S. patent application Ser. No. 13/072,047, filed Mar. 25,2011, which claims priority to U.S. Provisional Patent Application No.61/318,077, filed Mar. 26, 2010, each of which is incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention provides methods and materials related to thedetection of colorectal neoplasm-specific markers in or associated witha subject's stool sample. In particular, the present invention providesmethods and materials for identifying mammals having a colorectalneoplasm by detecting the presence of exfoliated epithelial markers(e.g., human DNA, tumor assoicated gene alterations, tumor associatedproteins) and blood markers (e.g., homoglobin, serum proteins) in astool sample obtained from the mammal.

BACKGROUND OF THE INVENTION

Colorectal cancer remains a leading cause of death among the types ofcancer (see, e.g., Jemal A, et al., CA Cancer J Clin. 2007, 57:43-66;herein incorporated by reference in its entirety). Although screeningreduces colorectal cancer mortality (see, e.g., Mandel J S, et al., NEngl J Med. 1993, 328:1365-71; Hardcastle J D, et al., Lancet. 1996,348:1472-7; Kronborg O, et al., Scand J Gastroenterol. 2004, 39:846-51;Winawer S J, et al., J Natl Cancer Inst. 1993, 85:1311-8; Singh H, etal., JAMA. 2006, 295:2366-73; each herein incorporated by reference intheir entireties), observed reductions have been modest (see, e.g.,Singh H, et al., JAMA. 2006; 295, 2366-73; Heresbach D, et al., Eur JGastroenterol Hepatol. 2006, 18:427-33; each herein incorporated byreference in their entireties) and more than one half of adults in theUnited States have not received screening (see, e.g., Meissner H I,Cancer Epidemiol Biomarkers Prev. 2006, 15:389-94; herein incorporatedby reference in its entirety). More accurate, user-friendly, and widelydistributable tools to improve screening effectiveness, acceptability,and access are needed.

SUMMARY

Fecal long DNA originate from either the exfoliation of dysplastic cellsor from the luminal hemorrhage or exudation of leukocytes. Previousreports have speculated that long DNA and occult blood in stool resultedfrom bleeding, and as such, speculated that fecal long DNA testing mightbe an alternative of fecal occult blood testing (see, e.g., de Kok J B,Clin Chem. 2003; 49:2112-2113; herein incorporated by reference in itsentirety). Experiments conducted during preparation of the embodimentsfor the present invention, however, demonstrated that long DNA was acontinuously measurable marker in stool, and occult blood was detectableonly intermittently (see, e.g., Osborn N K, et al., Gastroenterology2005; 128:192-206, Ahlquist D A, Cancer 1989; 63:1826-1830; each hereinincorporated by reference in their entireties) thereby indicating thatlong DNA and occult blood were from different sources and, as such, werecomplementary markers for CRC detection. Experiments furtherdemonstrated that long DNA and occult blood in stools provide asensitive approach for the detection of colorectal cancer.

Accordingly, in certain embodiments, the present invention providesmethods for detecting the presence of a colorectal neoplasm in a mammal.In some embodiments, the methods involve obtaining a stool sample from amammal, detecting the presence or absence of one or more exfoliatedepithelial markers specific for a colorectal neoplasm in or associatedwith the stool sample, and detecting the presence or absence of one ormore fecal occult blood markers (e.g., specific for a colorectalneoplasm) in the stool sample. In some embodiments, detection of thepresence of one or more exfoliated epithelial markers in the stoolsample in combination with the presence of one or more fecal occultblood markers in the stool sample is indicative of a colorectal neoplasmin the mammal.

In some embodiments, the mammal is a human. In some embodiments, thecolorectal neoplasm is premalignant. In some embodiments, the colorectalneoplasm is malignant.

The methods are not limited to particular exfoliated epithelial markersspecific for a colorectal neoplasm. In some embodiments, the one or morecolorectal neoplasm-specific nucleic acid markers include, for example,a gene having a point mutation, a gene reflecting microsatteliteinstability, a gene having aberrant methylation, and/or long DNA.

In some embodiments, a gene having a point mutation includes, but is notlimited to, K-ras, APC (see, e.g., U.S. Pat. Nos. 5,352,775, 5,648,212,5,691,454, 5,783,666, RE36,713, 6,114,124, 6,413,727, and RE38,916; eachherein incorporated by reference in their entireties), melanoma antigengene, p53 (see, e.g., U.S. Pat. Nos. 5,362,623, 5,527,676, 5,955,263,6,090,566, 6,245,515, 6,677,312, 6,800,617, 7,087,583, 7,267,955; eachherein incorporated by reference in their entireties), BRAF, BAT26 andPIK3CA (see, e.g., U.S. patent application Ser. No. 10/591,347; hereinincorporated by reference in its entirety). In some embodiments, a genereflecting microsatellite instability is BAT26. In some embodiments, agene having aberrant methylation includes, but is not limited to, bmp-3,bmp-4, SFRP2, vimentin (see, e.g., U.S. Pat. No. 7,485,402; hereinincorporated by reference in its entirety), septin9, ALX4, EYA4, TFPI2,NDRG4, HLTF (see, e.g., U.S. Pat. No. 7,432,050; herein incorporated byreference in its entirety), and FOXE1. In some embodiments, the long DNAis, for example, greater than 250 base pairs in length, greater than 300base pairs in length, greater than 400 base pairs in length, greaterthan 500 base pairs in length, and/or greater than 1000 base pairs inlength.

The methods are not limited to particular fecal occult blood markersspecific for a colorectal neoplasm. In some embodiments, fecal occultblood markers specific for a colorectal neoplasm include, but are notlimited to, hemoglobin, alpha-defensin, calprotectin, al-antitrypsin,albumin, MCM2, transferrin, lactoferrin, and lysozyme.

In some embodiments, the exfoliated epithelial marker specific forcolorectal neoplasm is long DNA, and the fecal occult blood markerspecific for colorectal neoplasm is hemoglobin.

In some embodiments wherein a colorectal neoplasm is detected,additional techniques are performed to characterise the colorectalneoplasm (e.g., to characterize the colorectal neoplasm as malignant orpremalignant) (e.g., to characterize the colorectal neoplasm within aparticular stage of colorectal cancer).

In certain embodiments, the present invention provides kits fordetecting the presence of a colorectal neoplasm in a mammal. In someembodiments, such kits include reagents useful, sufficient, or necessaryfor detecting and/or characterizing one or more exfoliated epithelialmarkers specific for a colorectal neoplasm, and reagents useful,sufficient, or necessary for detecting and/or characterizing one or morefecal occult blood markers specific for a colorectal neoplasm. In someembodiments, the kits contain the reagents necessary to performreal-time Alu PCR. In some embodiments, the kits contain the reagentsnecessary to perform the heme porphyrin test HemoQuant. In someembodiments, the kits contain the ingredients and reagents necessary toobtain and store a stool sample from a subject.

In certain embodiments, the present invention provides methods formonitoring the treatment of colorectal cancer. For example, in someembodiments, the methods may be performed immediately before, duringand/or after a treatment to monitor treatment success.

In some embodiments, the methods are performed at intervals on diseasefree patients to insure or monitor treatment success.

In certain embodiments, the present invention provides methods forobtaining a subject's risk profile for developing colorectal cancer. Insome embodiments, such methods involve obtaining a stool sample from asubject (e.g., a human at risk for developing colorectal cancer; a humanundergoing a routine physical examination), detecting the presence orabsence of one or more exfoliated epithelial markers specific for acolorectal neoplasm in or associated with the stool sample, detectingthe presence or absence of one or more fecal occult blood markers (e.g.,specific for a colorectal neoplasm) in or associated with the stoolsample, and generating a risk profile for developing colorectal cancerbased upon the detected presence or absence of the exfoliated epithelialmarkers and fecal occult blood markers. For example, in someembodiments, a generated risk profile will change depending uponspecific exfoliated epithelial markers and fecal occult blood markersdetected as present or absent. The present invention is not limited to aparticular manner of generating the risk profile. In some embodiments, aprocessor (e.g., computer) is used to generate such a risk profile. Insome embodiments, the processor uses an algorithm (e.g., software)specific for interpreting the presence and absence of specificexfoliated epithelial markers and fecal occult blood markers asdetermined with the methods of the present invention. In someembodiments, the presence and absence of specific exfoliated epithelialmarkers and fecal occult blood markers as determined with the methods ofthe present invention are inputed into such an algorithm, and the riskprofile is reported based upon a comparison of such input withestablished norms (e.g., established norm for pre-cancerous condition,established norm for various risk levels for developing colorectalcancer, established norm for subjects diagnosed with various stages ofcolorectal cancer). In some embodiments, the risk profile indicates asubject's risk for developing colorectal cancer or a subject's risk forre-developing colorectal cancer. In some embodiments, the risk profileindicates a subject to be, for example, at a very low, a low, amoderate, a high, and a very high chance of developing or re-developingcolorectal cancer. In some embodiments, the risk profile indicates riskbased on a population average at a desired level of specificity (e.g.,90%). In some embodiments, a health care provider (e.g., an oncologist)will use such a risk profile in determining a course of treatment orintervention (e.g., colonoscopy, wait and see, referral to anoncologist, referral to a surgeon, etc.).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B shows the levels of long DNA and occult blood in stools frompatients with CRCs or advanced adenomas and from normal individualsdisplayed in log scale. Each circle represents one stool sample.

FIG. 2 shows correlation of long DNA with occult blood levels in stoolsamples displayed in a scatter plot graph. A black trend is drawn toshow that fecal long DNA is not correlated with occult blood levels(R²=0.0001). Because zero cannot be plotted in log scale, long DNA levelplus 1 and occult blood level plus 0.1 are displayed here.

FIG. 3A-B shows receiver operating curves for stool long DNA and occultblood levels in patients with colorectal cancers or advanced adenomasversus normal controls. For cancers versus normal controls, AUC valueswere 0.82, 0.78, and 0.90 for fecal long DNA, occult blood, andcombination testing, respectively; for advanced adenomas versus normalcontrols, AUC values were 0.72, 0.50, and 0.72 for fecal DNA, occultblood, and combination testing, respectively. A, B, and C representreceiver operating curves for fecal long DNA, occult blood, andcombination testing, respectively.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below

As used herein, the term “colorectal cancer” is meant to include thewell-accepted medical definition that defines colorectal cancer as amedical condition characterized by cancer of cells of the intestinaltract below the small intestine (e.g., the large intestine (colon),including the cecum, ascending colon, transverse colon, descendingcolon, and sigmoid colon, and rectum). Additionally, as used herein, theterm “colorectal cancer” is meant to further include medical conditionswhich are characterized by cancer of cells of the duodenum and smallintestine (jejunum and ileum).

As used herein, the term “metastasis” is meant to refer to the processin which cancer cells originating in one organ or part of the bodyrelocate to another part of the body and continue to replicate.Metastasized cells subsequently form tumors which may furthermetastasize. Metastasis thus refers to the spread of cancer from thepart of the body where it originally occurs to other parts of the body.As used herein, the term “metastasized colorectal cancer cells” is meantto refer to colorectal cancer cells which have metastasized; colorectalcancer cells localized in a part of the body other than the duodenum,small intestine (jejunum and ileum), large intestine (colon), includingthe cecum, ascending colon, transverse colon, descending colon, andsigmoid colon, and rectum.

As used herein, “an individual is suspected of being susceptible tometastasized colorectal cancer” is meant to refer to an individual whois at an above-average risk of developing metastasized colorectalcancer. Examples of individuals at a particular risk of developingmetastasized colorectal cancer are those whose family medical historyindicates above average incidence of colorectal cancer among familymembers and/or those who have already developed colorectal cancer andhave been effectively treated who therefore face a risk of relapse andrecurrence. Other factors which may contribute to an above-average riskof developing metastasized colorectal cancer which would thereby lead tothe classification of an individual as being suspected of beingsusceptible to metastasized colorectal cancer may be based upon anindividual's specific genetic, medical and/or behavioral background andcharacteristics.

The term “neoplasm” as used herein refers to any new and abnormal growthof tissue. Thus, a neoplasm can be a premalignant neoplasm or amalignant neoplasm. The term “neoplasm-specific marker” refers to anybiological material that can be used to indicate the presence of aneoplasm. Examples of biological materials include, without limitation,nucleic acids, polypeptides, carbohydrates, fatty acids, cellularcomponents (e.g., cell membranes and mitochondria), and whole cells. Theterm “colorectal neoplasm-specific marker” refers to any biologicalmaterial that can be used to indicate the presence of a colorectalneoplasm (e.g., a premalignant colorectal neoplasm; a malignantcolorectal neoplasm). Examples of colorectal neoplasm-specific markersinclude, but are not limited to, exfoliated epithelial markes (e.g.,bmp-3, bmp-4, SFRP2, vimentin, septin9, ALX4, EYA4, TFPI2, NDRG4, FOXE1,long DNA, BAT-26, K-ras, APC, melanoma antigen gene, p53, BRAF, andPIK3CA) and fecal occult blood markers (e.g., hemoglobin,alpha-defensin, calprotectin, α1-antitrypsin, albumin, MCM2,transferrin, lactoferrin, and lysozyme).

As used herein, the term “adenoma” refers to a benign tumor of glandularorigin. Although these growths are benign, over time they may progressto become malignant. As used herein the term “colorectal adenoma” refersto a benign colorectal tumor in which the cells form recognizableglandular structures or in which the cells are clearly derived fromglandular epithelium.

As used herein, the term “amplicon” refers to a nucleic acid generatedusing primer pairs. The amplicon is typically single-stranded DNA (e.g.,the result of asymmetric amplification), however, it may be RNA ordsDNA.

The term “amplifying” or “amplification” in the context of nucleic acidsrefers to the production of multiple copies of a polynucleotide, or aportion of the polynucleotide, typically starting from a small amount ofthe polynucleotide (e.g., a single polynucleotide molecule), where theamplification products or amplicons are generally detectable.Amplification of polynucleotides encompasses a variety of chemical andenzymatic processes. The generation of multiple DNA copies from one or afew copies of a target or template DNA molecule during a polymerasechain reaction (PCR) or a ligase chain reaction (LCR; see, e.g., U.S.Pat. No. 5,494,810; herein incorporated by reference in its entirety)are forms of amplification. Additional types of amplification include,but are not limited to, allele-specific PCR (see, e.g., U.S. Pat. No.5,639,611; herein incorporated by reference in its entirety), assemblyPCR (see, e.g., U.S. Pat. No. 5,965,408; herein incorporated byreference in its entirety), helicase-dependent amplification (see, e.g.,U.S. Pat. No. 7,662,594; herein incorporated by reference in itsentirety), Hot-start PCR (see, e.g., U.S. Pat. Nos. 5,773,258 and5,338,671; each herein incorporated by reference in their entireties),intersequence-specfic PCR, inverse PCR (see, e.g., Triglia, et al.(1988) Nucleic Acids Res., 16:8186; herein incorporated by reference inits entirety), ligation-mediated PCR (see, e.g., Guilfoyle, R. et al.,Nucleic Acids Research, 25:1854-1858 (1997); U.S. Pat. No. 5,508,169;each of which are herein incorporated by reference in their entireties),methylation-specific PCR (see, e.g., Herman, et al., (1996) PNAS 93(13)9821-9826; herein incorporated by reference in its entirety), miniprimerPCR, multiplex ligation-dependent probe amplification (see, e.g.,Schouten, et al., (2002) Nucleic Acids Research 30(12): e57; hereinincorporated by reference in its entirety), multiplex PCR (see, e.g.,Chamberlain, et al., (1988) Nucleic Acids Research 16(23) 11141-11156;Ballabio, et al., (1990) Human Genetics 84(6) 571-573; Hayden, et al.,(2008) BMC Genetics 9:80; each of which are herein incorporated byreference in their entireties), nested PCR, overlap-extension PCR (see,e.g., Higuchi, et al., (1988) Nucleic Acids Research 16(15) 7351-7367;herein incorporated by reference in its entirety), real time PCR (see,e.g., Higuchi, etl al., (1992) Biotechnology 10:413-417; Higuchi, etal., (1993) Biotechnology 11:1026-1030; each of which are hereinincorporated by reference in their entireties), reverse transcriptionPCR (see, e.g., Bustin, S. A. (2000) J. Molecular Endocrinology25:169-193; herein incorporated by reference in its entirety), solidphase PCR, thermal asymmetric interlaced PCR, and Touchdown PCR (see,e.g., Don, et al., Nucleic Acids Research (1991) 19(14) 4008; Roux, K.(1994) Biotechniques 16(5) 812-814; Hecker, et al., (1996) Biotechniques20(3) 478-485; each of which are herein incorporated by reference intheir entireties). Polynucleotide amplification also can be accomplishedusing digital PCR (see, e.g., Kalinina, et al., Nucleic Acids Research.25; 1999-2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA.96; 9236-41, (1999); International Patent Publication No. WO05023091A2;US Patent Application Publication No. 20070202525; each of which areincorporated herein by reference in their entireties).

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence“5′-A-G-T-3′,” is complementary to the sequence “3′-T-C-A-5′.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods that depend uponbinding between nucleic acids.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, that is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product that is complementary to a nucleic acid strand isinduced (e.g., in the presence of nucleotides and an inducing agent suchas a biocatalyst (e.g., a DNA polymerase or the like) and at a suitabletemperature and pH). The primer is typically single stranded for maximumefficiency in amplification, but may alternatively be double stranded.If double stranded, the primer is generally first treated to separateits strands before being used to prepare extension products. In someembodiments, the primer is an oligodeoxyribonucleotide. The primer issufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer and theuse of the method. In certain embodiments, the primer is a captureprimer.

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4 acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

As used herein, the term “nucleobase” is synonymous with other terms inuse in the art including “nucleotide,” “deoxynucleotide,” “nucleotideresidue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” ordeoxynucleotide triphosphate (dNTP).

An “oligonucleotide” refers to a nucleic acid that includes at least twonucleic acid monomer units (e.g., nucleotides), typically more thanthree monomer units, and more typically greater than ten monomer units.The exact size of an oligonucleotide generally depends on variousfactors, including the ultimate function or use of the oligonucleotide.To further illustrate, oligonucleotides are typically less than 200residues long (e.g., between 15 and 100), however, as used herein, theterm is also intended to encompass longer polynucleotide chains.Oligonucleotides are often referred to by their length. For example a 24residue oligonucleotide is referred to as a “24-mer”. Typically, thenucleoside monomers are linked by phosphodiester bonds or analogsthereof, including phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like, including associatedcounterions, e.g., H⁺, NH₄ ⁺, Na⁺, and the like, if such counterions arepresent. Further, oligonucleotides are typically single-stranded.Oligonucleotides are optionally prepared by any suitable method,including, but not limited to, isolation of an existing or naturalsequence, DNA replication or amplification, reverse transcription,cloning and restriction digestion of appropriate sequences, or directchemical synthesis by a method such as the phosphotriester method ofNarang et al. (1979) Meth Enzymol. 68: 90-99; the phosphodiester methodof Brown et al. (1979) Meth Enzymol. 68: 109-151; thediethylphosphoramidite method of Beaucage et al. (1981) TetrahedronLett. 22: 1859-1862; the triester method of Matteucci et al. (1981) J AmChem Soc. 103:3185-3191; automated synthesis methods; or the solidsupport method of U.S. Pat. No. 4,458,066, entitled “PROCESS FORPREPARING POLYNUCLEOTIDES,” issued Jul. 3, 1984 to Caruthers et al., orother methods known to those skilled in the art. All of these referencesare incorporated by reference.

A “sequence” of a biopolymer refers to the order and identity of monomerunits (e.g., nucleotides, etc.) in the biopolymer. The sequence (e.g.,base sequence) of a nucleic acid is typically read in the 5′ to 3′direction.

DETAILED DESCRIPTION OF THE INVENTION

An effective way to detect colorectal cancer (CRC) at early stages ispopulation screening. Colonoscopy and fecal occult blood testing (FOBT)are commonly used tools for CRC screening, but the adherent rates ofboth approaches are low due to the invasiveness and expense ofcolonoscopy and the low sensitivity of FOBT (see, e.g., Osborn N K, etal., Gastroenterology 2005; 128:192-206; Levin B, et al., CA Cancer JClin 2008; 58:130-160; each herein incorporated by reference in itsentirety). For example, fecal long DNA quantified with real-time Alu PCRis a simple approach for CRC detection, but its sensitivity is lessoptimal when assayed alone (see, e.g., Zou H, et al., Cancer EpidemiolBiomarkers Prev 2006; 15:1115-1119; herein incorporated by reference inits entirety).

Fecal long DNA originate from either the exfoliation of dysplastic cellsor from the luminal hemorrhage or exudation of leukocytes. Previousreports have speculated that long DNA and occult blood in stool resultedfrom bleeding, and as such, speculated that fecal long DNA testing mightbe an alternative of fecal occult blood testing (see, e.g., de Kok J B,Clin Chem. 2003; 49:2112-2113; herein incorporated by reference in itsentirety). Experiments conducted during preparation of the embodimentsfor the present invention, however, demonstrated that long DNA was acontinuously measurable marker in stool, and occult blood was detectableonly intermittently (see, e.g., Osborn N K, et al., Gastroenterology2005; 128:192-206, Ahlquist D A, Cancer 1989; 63:1826-1830; each hereinincorporated by reference in their entireties) thereby indicating thatlong DNA and occult blood were from different sources and, as such, werecomplementary markers for CRC detection. Experiments furtherdemonstrated that long DNA and occult blood in stools provide asensitive approach for the detection of colorectal cancer.

Ideally, approaches for screening colorectal cancer should be accurate,user friendly, and inexpensive. Both fecal long DNA and occult bloodtestings are simple non-invasive approaches, but their sensitivities forCRC detection are less optimal when assayed alone (see, e.g., Zou H, etal., Cancer Epidemiol Biomarkers Prev 2006; 15:1115-1119; Mandel J S, etal., N Engl J Med 1993; 328:1365-1371; Kronborg 0, et al., Lancet 1996;348:1467-1471; Hardcastle J D, et al., Lancet 1996; 348:1472-1477;Ahlquist D A, et al., JAMA 1993; 269:1262-1267; Imperiale T F, et al., NEngl J Med 2004; 351:2704-2714; Ahlquist D A, Ann Intern Med 2008;149:441-450; Ahlquist D A, Gastroenterology 2000; 119:1219-1227; eachherein incorporated by reference in their entireties). As experimentsconducted during preparation of the embodiments for the presentinvention demonstrated that stool based long DNA and fecal occult bloodare complementary markers originating from different sources, thepresent invention, in some embodiments, provides a quantitative assaycombining exfoliated epithelial markers (e.g., long DNA) and fecaloccult blood markers (e.g., hemoglobin) yielding an inexpensive approachfor sensitive detection of CRC.

Accordingly, the present invention provides methods and materialsrelated to the detection of colorectal cancer-specific markers in asubject's stool sample. In particular, the present invention providesmethods and materials for identifying mammals having a colorectal cancerby detecting the presence of exfoliated epithelial markers (e.g., humanlong DNA, tumor assoicated gene alterations, tumor associated proteins)and fecal occult blood markers (e.g., hemoglobin, serum proteins) in astool sample obtained from the mammal.

While the present invention exemplifies several markers specific fordetecting colorectal cancer, any marker that is correlated with thepresence or absence of colorectal cancer may be used. A marker, as usedherein, includes, for example, any proteinaceous molecule (orcorresponding gene) whose production or lack of production ischaracteristic of a colorectal cancer cell. Depending on the particularset of markers employed in a given analysis, the statistical analysiswill vary. For example, where a particular combination of markers ishighly specific for colorectal cancer, the statistical significance of apositive result will be high. It may be, however, that such specificityis achieved at the cost of sensitivity (e.g., a negative result mayoccur even in the presence of colorectal cancer). By the same token, adifferent combination may be very sensitive (e.g., few false negatives,but has a lower specificity).

Particular combinations of markers may be used that show optimalfunction with different ethnic groups or sex, different geographicdistributions, different stages of disease, different degrees ofspecificity or different degrees of sensitivity. Particular combinationsmay also be developed which are particularly sensitive to the effect oftherapeutic regimens on disease progression. Subjects may be monitoredafter a therapy and/or course of action to determine the effectivenessof that specific therapy and/or course of action.

As noted, experiments conducted during the course of developingembodiments for the present invention determined an enhanced method andsystem for detecting colorectal cancer through detecting and assessingboth the presence or absence of exfoliated epithelial markers (e.g.,human DNA, tumor assoicated gene alterations, tumor associated proteins)and fecal occult blood markers (e.g., hemoglobin, serum proteins) in astool sample obtained from the mammal.

The present invention is not limited to detecting particular exfoliatedepithelial markers specific for detecting colorectal cancer. In someembodiments, the exfoliated epithelial markers specific for detectingcolorectal cancer include nucleic acid. Examples of colorectalcancer-specific nucleic acid markers include, without limitation,nucleic acid having a point mutation, nucleic acid that reflectsmicrosatellite instability, nucleic acid having aberrrant methylation,and long DNA.

Nucleic acid having a point mutation can encode a polypeptide orregulate the expression of a polypeptide (e.g., promoters, enhancers,and silencers). Examples of nucleic acid that can contain a pointmutation indicative of a colorectal cancer include, without limitation,the genes for K-ras, APC (adenomatous polyposis coli), melanoma antigengene, p53, BRAF, BAT26, and PIK3CA.

Nucleic acid that reflects microsatellite instability can be used toindicate the presence of colorectal cancer. Briefly, nucleic acid thatreflects microsatellite instability can be identified as describedelsewhere (see, e.g., Samowitz et al., Am. J. Path., 154:1637-1641(1999); Hoang et al., Cancer Res., 57:300-303 (1997); each hereinincorporated by reference in its entirety). An example of nucleic acidthat can reflect microsatellite instability indicative of a colorectalneoplasm includes, without limitation, the gene for BAT-26.

Nucleic acid having aberrant methylation can be used to indicate thepresence of colorectal cancer (see, e.g., Muller, et al., Lancet 2004363:1283-1285; herein incorporated by reference in its entirety).Examples of nucleic acid having aberrant methylation indicative ofcolorectal cancer include, but are not limited to, bmp-3, bmp-4, SFRP2,vimentin, septin9, ALX4, EYA4, TFPI2, NDRG4, and FOXE1.

The presence of long DNA in a stool sample is indicative of colorectalcancer. Long DNA is a marker for non-apoptotic cells. Typically, cellsshed from normal mucosa are apoptotic, while those shed from colorectalneoplasms are non-apoptotic. As described herein, long DNA can be usedas a colorectal cancer-specific marker for subjects having colorectalcancer. One hallmark of apoptosis is the autodigestion or cleavage ofDNA into “short” fragments of about 180 base-pairs. The detection of“long” DNA (e.g., DNA greater than about 200 base-pairs) in a stoolsample can indicate the presence of non-apoptotic cells of neoplasticlineage derived from a colorectal neoplasm. The term “long DNA” as usedherein refers to DNA greater than about 200 base-pairs (e.g., greaterthan about 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1250,1500, 1750, 2000, or 2500 base-pairs).

The present invention is not limited to a particular method fordetecting and/or quantifying long DNA within a subject's stool sample.In some embodiments, real-time Alu PCR is used for detecting and/orquantifying long DNA within a subject's stool sample. Real-time Alu PCRis a sensitive method for detecting non-apoptotic human DNA in stool asit targets abundant Alu repeats in human genome (see, e.g., Zou H, etal. Cancer Epidemiol Biomarkers Prev 2006, 15:1115-1119; hereinincorporated by reference in its entirety). Alu sequences embody thelargest family of middle repetitive DNA sequences in human genome (see,e.g., Kariya Y, et al., Gene 1987, 53:1-10; herein incorporated byreference in its entirety). An estimated half million Alu copies arepresent per haploid human genome (see, e.g., Kariya Y, et al., Gene1987, 53:1-10; herein incorporated by reference in its entirety).Accordingly, as Alu sequences are so abundantly distributed throughoutthe genome and specific to the genomes primates, real-time Alu PCRamplifies DNA sequences longer than 200 bp within these 300-bp repeats(see, e.g., Kariya Y, et al., Gene 1987, 53:1-10; herein incorporated byreference in its entirety) thereby providing a genome-wide approach toquantify human long DNA in stool (see, e.g., Zou H, et al. CancerEpidemiol Biomarkers Prev 2006, 15:1115-1119; herein incorporated byreference in its entirety).

The present invention is not limited to detecting particular fecaloccult blood markers specific for colorectal cancer. In someembodiments, the fecal occult blood markers specific for detectingcolorectal neoplasm include, without limitation, hemoglobin,alpha-defensin, calprotectin, α1-antitrypsin, albumin, MCM2,transferrin, lactoferrin, and lysozyme.

Any fecal occult blood testing method can be used to detect colorectalcancer-specific fecal occult blood markers. For example, antibodiesspecific for a polypeptide marker can be used in an immunoassay (e.g.,ELISA) to detect the presence or absence of the polypeptide in a stoolsample that is indicative of the presence of colorectal cancer. Toaccurately evaluate bleeding in the digestive tract, it is importantthat fecal occult blood tests target analytes that are stable during theenteric transit. Available data indicate that both guaiac andimmunochemical FOBTs are insensitive for the detection of bleeding fromproximal colon (see, e.g., Imperiale, et al., N Engl J Med 2004,351:2704-2714; Ahlquist, et al., Ann Intern Med 2008, 149:441-450;Harewood, et al., Mayo Clin Proc 2002, 77:23-28; Allison, et al., J NatlCancer Inst 2007, 99:1462-1470; each herein incorporated by reference intheir entireties). In contrast, the heme porphyrin test HemoQuant issensitive for both proximal and distal sources of occult bleeding (see,e.g., Harewood, et al., Mayo Clin Proc 2002, 77:23-28; Ahlquist, et al.,N Engl J Med 1985, 312:1422-1428; Harewood, et al., Dig Dis. 2000,18(2):75-82; each herein incorporated by reference in their entireties).Accordingly, in some embodiments, the heme porphyrin test HemoQuant isused for detecting and/or quantifying the colorectal cancer-specificfecal occult blood marker hemoglobin.

The present invention is not limited to a particular combination ofexfoliated epithelial markers and fecal occult blood markers in thedetection of colorectal cancer in a subject. In some embodiments, anyone or more exfoliated epithelial markers are used (e.g., bmp-3, bmp-4,SFRP2, vimentin, septin9, ALX4, EYA4, TFPI2, NDRG4, FOXE1, long DNA,BAT-26, K-ras, APC, melanoma antigen gene, p53, BRAF, and PIK3CA). Insome embodiments, any one or more fecal occult blood markers are used(e.g., hemoglobin, alpha-defensin, calprotectin, α1-antitrypsin,albumin, MCM2, transferrin, lactoferrin, and lysozyme).

It is noted that a single stool sample can be analyzed for onecolorectal neoplasm-specific marker or for multiple colorectalneoplasm-specific markers. For example, a stool sample can be analyzedusing assays that detect a panel of different colorectalneoplasm-specific markers. In addition, multiple stool samples can becollected for a single mammal and analyzed as described herein. Indeed,U.S. Pat. Nos. 5,670,325, 5,741,650, 5,928,870, 5,952,178, and6,020,137, each herein incorporated by reference in their entireties,for example, describe various methods that can be used to prepare andanalyze stool samples. In some embodiments, a stool sample is split intofirst and second portions, where the first portion undergoes analysisfor exfoliated epithelial markers and the second portion undergoesanalysis for fecal occult blood markers. In some embodiments, the stoolsample undergoes one or more preprocessing steps before being split intoportions.

The present invention is not limited to a particular manner of detectingnucleic acid markers corresponding to colorectal neoplasm from a stoolsample. In some embodiments, nucleic acid is amplified. Generally,nucleic acid used as template for amplification is isolated from cellscontained in the biological sample according to standard methodologies(see, e.g., Sambrook, J., et al., Fritsch, E. F., Maniatis, T. (ed.).MOLECULAR CLONING. Cold Spring Harbor Lab. Press, Cold Spring Harbor,N.Y. (1989); herein incorporated by reference in its entirety). Thenucleic acid may be genomic DNA or fractionated or whole cell RNA. WhereRNA is used, it may be desired to convert the RNA to a complementarycDNA. In a preferred embodiment, the RNA is whole cell RNA and is useddirectly as the template for amplification. Pairs of primers thatselectively hybridize to genes corresponding to specific markers arecontacted with the isolated nucleic acid under conditions that permitselective hybridization. Once hybridized, the nucleic acid primercomplex is contacted with one or more enzymes that facilitatetemplate-dependent nucleic acid synthesis. Multiple rounds ofamplification, also referred to as “cycles,” are conducted until asufficient amount of amplification product is produced. Next, theamplification product is detected. In some applications, the detectionmay be performed by visual means. Alternatively, the detection mayinvolve indirect identification of the product via chemiluminescence,radioactive scintigraphy of incorporated radio label or fluorescentlabel or even via a system using electrical or thermal impulse signals.Generally, the foregoing process is conducted at least twice on a givensample using at least two different primer pairs specific for twodifferent specific markers. Following detection, in some embodiments,the results seen in a given subject are compared with a statisticallysignificant reference group of subjects diagnosed as not havingcolorectal cancer.

The term primer, as defined herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty base pairs in length, but longer sequences can beemployed. Primers may be provided in double-stranded or single-strandedform, although the single-stranded form is preferred.

In most cases, it will be preferable to synthesize desiredoligonucleotides. Suitable primers can be synthesized using commercialsynthesizers using methods well known to those of ordinary skill in theart. Where double-stranded primers are desired, synthesis ofcomplementary primers is performed separately and the primers mixedunder conditions permitting their hybridization.

Selection of primers is based on a variety of different factors,depending on the method of amplification and the specific markerinvolved. For example, the choice of primer will determine thespecificity of the amplification reaction. The primer needs to besufficiently long to specifically hybridize to the marker nucleic acidand allow synthesis of amplification products in the presence of thepolymerization agent and under appropriate temperature conditions.Shorter primer molecules generally require cooler temperatures to formsufficiently stable hybrid complexes with the marker nucleic acid andmay be more susceptible to non-specific hybridization and amplification.

Primer sequences do not need to correspond exactly to the specificmarker sequence. Non-complementary nucleotide fragments may be attachedto the 5′ end of the primer with the remainder of the primer sequencebeing complementary to the template. Alternatively, non-complementarybases can be interspersed into the primer, provided that the primersequence has sufficient complementarily, in particular at the 3′ end,with the template for annealing to occur and allow synthesis of acomplementary DNA strand.

In some embodiments, primers may be designed to hybridize to specificregions of the marker nucleic acid sequence. For example, GC richregions are favored as they form stronger hybridization complexes thanAT rich regions. In another example, primers are designed, solely, tohybridize to a pair of exon sequences, with at least one intron inbetween. This allows for the activity of a marker gene to be detected asopposed to its presence by minimizing background amplification of thegenomic sequences and readily distinguishes the target amplification bysize.

Primers also may be designed to amplify a particular segment of markernucleic acid that encodes restriction sites. A restriction site in thefinal amplification product would enable digestion at that particularsite by the relevant restriction enzyme to produce two products of aspecific size. Any restriction enzyme may be utilized in this aspect.This added refinement to the amplification process may be necessary whenamplifying a marker nucleic acid sequence with close sequence similarityto other nucleic acids. Alternatively, it may be used as an addedconfirmation of the specificity of the amplification product.

A number of template dependent processes are available to amplify themarker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (PCR) (see,e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, and Innis et al.,PCR Protocols, Academic Press, Inc., San Diego, Calif. (1990); eachincorporated herein by reference in their entireties). Briefly, in PCR,two primer sequences are prepared which are complementary to regions onopposite complementary strands of the marker sequence. An excess ofdeoxynucleoside triphosphates are added to a reaction mixture along witha DNA polymerase, e.g., Taq polymerase. If the marker sequence ispresent in a sample, the primers will bind to the marker and thepolymerase will cause the primers to be extended along the markersequence by adding on nucleotides. By raising and lowering thetemperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated. In some embodiments, a reverse transcriptase PCR amplificationprocedure is performed in order to quantify the amount of mRNAamplified. Methods of reverse transcribing RNA into cDNA are well known(see, e.g., Sambrook, J., et al., Fritsch, E. F., Maniatis, T. (ed.).MOLECULAR CLONING. Cold Spring Harbor Lab. Press, Cold Spring Harbor,N.Y. (1989); herein incorporated by reference in its entirety).Alternatively, methods for reverse transcription utilize thermostableDNA polymerases (see, e.g., WO 90/07641; herein incorporated byreference in its entirety).

The present invention is not limited to a particular PCR technique.Examples of PCR include, but are not limited to, standard PCR,allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR,helicase-dependent amplification, Hot-start PCR, interseqeunce-specificPCR, inverse PCR, ligation-mediated PCR, methylation-specific PCR,miniprimer PCR, multiplex ligation-dependent probe amplification, nestedPCR, overlap-extension PCR, real-time PCR, reverse transcription PCR,solid phase PCR, thermal asymmetric interlaced PCR, and Touchdown PCR.

Another method for amplification is the ligase chain reaction (“LCR”)(see, e.g., U.S. Pat. Nos. 4,883,750 and 5,494,810; herein incorporatedby reference in its entirety). In LCR, two complementary probe pairs areprepared, and in the presence of the marker sequence, each pair willbind to opposite complementary strands of the marker such that theyabut. In the presence of a ligase, the two probe pairs will link to forma single unit. By temperature cycling, as in PCR, bound ligated unitsdissociate from the marker and then serve as “target sequences” forligation of excess probe pairs.

Following amplification, it may be desirable to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification occurred. In someembodiments, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (see, e.g., Sambrook, J., et al., Fritsch, E. F., Maniatis, T.(ed.). MOLECULAR CLONING. Cold Spring Harbor Lab. Press, Cold SpringHarbor, N.Y. (1989); herein incorporated by reference in its entirety).

Alternatively, chromatographic techniques may be employed to effectseparation. There are many kinds of chromatography which may be used inthe present invention: adsorption, partition, ion-exchange and molecularsieve, and many specialized techniques for using them including column,paper, thin-layer and gas chromatography (see, e.g., Freifelder, D.Physical Biochemistry Applications to Biochemistry and MolecularBiology. 2nd ed. Wm. Freeman & Co., New York, N.Y. 1982; incorporatedherein by reference in its entirety).

Amplification products must be visualized in order to confirmamplification of the marker sequences. One typical visualization methodinvolves staining of a gel with ethidium bromide and visualization underUV light. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theamplification products can then be exposed to x-ray film or visualizedunder the appropriate stimulating spectra, following separation.

In some embodiments, visualization is achieved indirectly. For example,following separation of amplification products, a nucleic acid probe isbrought into contact with the amplified marker sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, where the other member of the binding paircarries a detectable moiety.

In some embodiments, detection is by Southern blotting and hybridizationwith a labeled probe. The techniques involved in Southern blotting arewell known to those of skill in the art and can be found in manystandard books on molecular protocols (see, e.g., Sambrook, J., et al.,Fritsch, E. F., Maniatis, T. (ed.). MOLECULAR CLONING. Cold SpringHarbor Lab. Press, Cold Spring Harbor, N.Y. (1989); herein incorporatedby reference in its entirety). Briefly, amplification products areseparated by gel electrophoresis. The gel is then contacted with amembrane, such as nitrocellulose, permitting transfer of the nucleicacid and non-covalent binding. Subsequently, the membrane is incubatedwith a chromophore conjugated probe that is capable of hybridizing witha target amplification product. Detection is by exposure of the membraneto x-ray film or ion-emitting detection devices.

In some embodiments, all the basic essential materials and reagentsrequired for detecting colorectal cancer through detecting both thepresence or absence of exfoliated epithelial markers (e.g., human DNA,tumor assoicated gene alterations, tumor associated proteins) and fecaloccult blood markers (e.g., hemoglobin, serum proteins) in a stoolsample obtained from the mammal are assembled together in a kit. Suchkits generally comprise, for example, reagents useful, sufficient, ornecessary for detecting and/or characterizing one or more exfoliatedepithelial markers specific for a colorectal neoplasm (e.g., bmp-3,bmp-4, SFRP2, vimentin, septin9, ALX4, EYA4, TFPI2, NDRG4, FOXE1, longDNA, BAT-26, K-ras, APC, melanoma antigen gene, p53, BRAF, and PIK3CA),and reagents useful, sufficient, or necessary for detecting and/orcharacterizing one or more fecal occult blood markers specific for acolorectal neoplasm (e.g., hemoglobin, alpha-defensin, calprotectin,α1-antitrypsin, albumin, MCM2, transferrin, lactoferrin, and lysozyme).In some embodiments, the kits contain enzymes suitable for amplifyingnucleic acids including various polymerases, deoxynucleotides andbuffers to provide the necessary reaction mixture for amplification. Insome embodiments, the kits contain reagents necessary to performReal-time Alu PCR. In some embodiments, the kits contain reagentsnecessary to perform the heme porphyrin test HemoQuant. In someembodiments, the kits of the present invention include a means forcontaining the reagents in close confinement for commercial sale suchas, e.g., injection or blow-molded plastic containers into which thedesired reagent are retained. Other containers suitable for conductingcertain steps of the disclosed methods also may be provided.

In some embodiments, the methods disclosed herein are useful inmonitoring the treatment of colorectal cancer. For example, in someembodiments, the methods may be performed immediately before, duringand/or after a treatment to monitor treatment success. In someembodiments, the methods are performed at intervals on disease freepatients to insure treatment success.

The present invention also provides a variety of computer-relatedembodiments. Specifically, in some embodiments the invention providescomputer programming for analyzing and comparing a pattern of colorectalneoplasm-specific marker (e.g., exfoliated epithelial markers and fecaloccult blood markers) detection results in a stool sample obtained froma subject to, for example, a library of such marker patterns known to beindicative of the presence or absence of a colorectal cancer, or aparticular stage or colorectal cancer.

In some embodiments, the present invention provides computer programmingfor analyzing and comparing a first and a second pattern of colorectalneoplasm-specific marker (e.g., exfoliated epithelial markers and fecaloccult blood markers) detection results from a stool sample taken at atleast two different time points. In some embodiments, the first patternmay be indicative of a pre-cancerous condition and/or low risk conditionfor colorectal cancer and/or progression from a pre-cancerous conditionto a cancerous condition. In such embodiments, the comparing providesfor monitoring of the progression of the condition from the first timepoint to the second time point.

In yet another embodiment, the invention provides computer programmingfor analyzing and comparing a pattern of colorectal neoplasm-specificmarker (e.g., exfoliated epithelial markers and fecal occult bloodmarkers) detection results from a stool sample to a library ofcolorectal neoplasm-specific marker patterns known to be indicative ofthe presence or absence of a colorectal cancer, wherein the comparingprovides, for example, a differential diagnosis between a benigncolorectal neoplasm, and an aggressively malignant colorectal neoplasm(e.g., the marker pattern provides for staging and/or grading of thecancerous condition).

The methods and systems described herein can be implemented in numerousways. In one embodiment, the methods involve use of a communicationsinfrastructure, for example the internet. Several embodiments of theinvention are discussed below. It is also to be understood that thepresent invention may be implemented in various forms of hardware,software, firmware, processors, or a combination thereof. The methodsand systems described herein can be implemented as a combination ofhardware and software. The software can be implemented as an applicationprogram tangibly embodied on a program storage device, or differentportions of the software implemented in the user's computing environment(e.g., as an applet) and on the reviewer's computing environment, wherethe reviewer may be located at a remote site (e.g., at a serviceprovider's facility).

For example, during or after data input by the user, portions of thedata processing can be performed in the user-side computing environment.For example, the user-side computing environment can be programmed toprovide for defined test codes to denote platform, carrier/diagnostictest, or both; processing of data using defined flags, and/or generationof flag configurations, where the responses are transmitted as processedor partially processed responses to the reviewer's computing environmentin the form of test code and flag configurations for subsequentexecution of one or more algorithms to provide a results and/or generatea report in the reviewer's computing environment.

The application program for executing the algorithms described hereinmay be uploaded to, and executed by, a machine comprising any suitablearchitecture. In general, the machine involves a computer platformhaving hardware such as one or more central processing units (CPU), arandom access memory (RAM), and input/output (I/O) interface(s). Thecomputer platform also includes an operating system and microinstructioncode. The various processes and functions described herein may either bepart of the microinstruction code or part of the application program (ora combination thereof) which is executed via the operating system. Inaddition, various other peripheral devices may be connected to thecomputer platform such as an additional data storage device and aprinting device.

As a computer system, the system generally includes a processor unit.The processor unit operates to receive information, which generallyincludes test data (e.g., specific gene products assayed), and testresult data (e.g., the pattern of colorectal neoplasm-specific marker(e.g., exfoliated epithelial markers and fecal occult blood markers)detection results from a stool sample). This information received can bestored at least temporarily in a database, and data analyzed incomparison to a library of marker patterns known to be indicative of thepresence or absence of a pre-cancerous condition, or known to beindicative of a stage and/or grade of colorectal cancer.

Part or all of the input and output data can also be sentelectronically; certain output data (e.g., reports) can be sentelectronically or telephonically (e.g., by facsimile, e.g., usingdevices such as fax back). Exemplary output receiving devices caninclude a display element, a printer, a facsimile device and the like.Electronic forms of transmission and/or display can include email,interactive television, and the like. In some embodiments, all or aportion of the input data and/or all or a portion of the output data(e.g., usually at least the library of the pattern of colorectalneoplasm-specific marker (e.g., exfoliated epithelial markers and fecaloccult blood markers) detection results known to be indicative of thepresence or absence of a pre-cancerous condition) are maintained on aserver for access, e.g., confidential access. The results may beaccessed or sent to professionals as desired.

A system for use in the methods described herein generally includes atleast one computer processor (e.g., where the method is carried out inits entirety at a single site) or at least two networked computerprocessors (e.g., where detected marker data for a stool sample obtainedfrom a subject is to be input by a user (e.g., a technician or someoneperforming the activity assays)) and transmitted to a remote site to asecond computer processor for analysis (e.g., where the pattern ofcolorectal neoplasm-specific marker (e.g., exfoliated epithelial markersand fecal occult blood markers) detection results is compared to alibrary of patterns known to be indicative of the presence or absence ofa pre-cancerous condition), where the first and second computerprocessors are connected by a network, e.g., via an intranet orinternet). The system can also include a user component(s) for input;and a reviewer component(s) for review of data, and generation ofreports, including detection of a pre-cancerous condition, stagingand/or grading of a colorectal neoplasm, or monitoring the progressionof a pre-cancerous condition or a colorectal neoplasm. Additionalcomponents of the system can include a server component(s); and adatabase(s) for storing data (e.g., as in a database of report elements,e.g., a library of marker patterns known to be indicative of thepresence or absence of a pre-cancerous condition and/or known to beindicative of a grade and/or a stage of a colorectal neoplasm, or arelational database (RDB) which can include data input by the user anddata output. The computer processors can be processors that aretypically found in personal desktop computers (e.g., IBM, Dell,Macintosh), portable computers, mainframes, minicomputers, or othercomputing devices.

The input components can be complete, stand-alone personal computersoffering a full range of power and features to run applications. Theuser component usually operates under any desired operating system andincludes a communication element (e.g., a modem or other hardware forconnecting to a network), one or more input devices (e.g., a keyboard,mouse, keypad, or other device used to transfer information orcommands), a storage element (e.g., a hard drive or othercomputer-readable, computer-writable storage medium), and a displayelement (e.g., a monitor, television, LCD, LED, or other display devicethat conveys information to the user). The user enters input commandsinto the computer processor through an input device. Generally, the userinterface is a graphical user interface (GUI) written for web browserapplications.

The server component(s) can be a personal computer, a minicomputer, or amainframe and offers data management, information sharing betweenclients, network administration and security. The application and anydatabases used can be on the same or different servers.

Other computing arrangements for the user and server(s), includingprocessing on a single machine such as a mainframe, a collection ofmachines, or other suitable configuration are contemplated. In general,the user and server machines work together to accomplish the processingof the present invention.

Where used, the database(s) is usually connected to the database servercomponent and can be any device which will hold data. For example, thedatabase can be any magnetic or optical storing device for a computer(e.g., CDROM, internal hard drive, tape drive). The database can belocated remote to the server component (with access via a network,modem, etc.) or locally to the server component.

Where used in the system and methods, the database can be a relationaldatabase that is organized and accessed according to relationshipsbetween data items. The relational database is generally composed of aplurality of tables (entities). The rows of a table represent records(collections of information about separate items) and the columnsrepresent fields (particular attributes of a record). In its simplestconception, the relational database is a collection of data entries that“relate” to each other through at least one common field.

Additional workstations equipped with computers and printers may be usedat point of service to enter data and, in some embodiments, generateappropriate reports, if desired. The computer(s) can have a shortcut(e.g., on the desktop) to launch the application to facilitateinitiation of data entry, transmission, analysis, report receipt, etc.as desired.

In certain embodiments, the present invention provides methods forobtaining a subject's risk profile for developing colorectal cancer. Insome embodiments, such methods involve obtaining a stool sample from asubject (e.g., a human at risk for developing colorectal cancer; a humanundergoing a routine physical examination), detecting the presence orabsence of one or more exfoliated epithelial markers specific for acolorectal neoplasm in or associated with the stool sample, detectingthe presence or absence of one or more fecal occult blood markers (e.g.,specific for a colorectal neoplasm) in or associated with the stoolsample, and generating a risk profile for developing colorectal cancerbased upon the detected presence or absence of the exfoliated epithelialmarkers and fecal occult blood markers. For example, in someembodiments, a generated risk profile will change depending uponspecific exfoliated epithelial markers and fecal occult blood markersdetected as present or absent. The present invention is not limited to aparticular manner of generating the risk profile. In some embodiments, aprocessor (e.g., computer) is used to generate such a risk profile. Insome embodiments, the processor uses an algorithm (e.g., software)specific for interpreting the presence and absence of specificexfoliated epithelial markers and fecal occult blood markers asdetermined with the methods of the present invention. In someembodiments, the presence and absence of specific exfoliated epithelialmarkers and fecal occult blood markers as determined with the methods ofthe present invention are inputed into such an algorithm, and the riskprofile is reported based upon a comparison of such input withestablished norms (e.g., established norm for pre-cancerous condition,established norm for various risk levels for developing colorectalcancer, established norm for subjects diagnosed with various stages ofcolorectal cancer). In some embodiments, the risk profile indicates asubject's risk for developing colorectal cancer or a subject's risk forre-developing colorectal cancer. In some embodiments, the risk profileindicates a subject to be, for example, a very low, a low, a moderate, ahigh, and a very high chance of developing or re-developing colorectalcancer. In some embodiments, the risk profile indicates risk based on apopulation average at a desire level of specificity (e.g., 90%). In someembodiments, a health care provider (e.g., an oncologist) will use sucha risk profile in determining a course of treatment or intervention(e.g., colonoscopy, wait and see, referral to an oncologist, referral toa surgeon, etc.).

EXAMPLES

The invention now being generally described, will be more readilyunderstood by reference to the following example, which is includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1

Subjects

A total of two hundred and one subjects, including 74 patients with CRC,27 with advanced adenoma (>1 cm), and 100 colonoscopically normalindividuals, were all recruited. Their demographic and clinicalcharacteristics are described in Table 1.

TABLE 1 Demographic and clinical characteristics of subjects ColorectalAdvanced cancer adenoma Normal control Number 74 27 100 Median Age(Range) 61 (40-87) yrs 67 (50-82) yrs 59 (28-81) yrs Sex (Male/Female)52/22 15/12 37/63 Site (Right/Left) 29/45 17/10 Stage (I/II/III/IV)13/16/27/18 Grade (1/2/3/4) or 0/4/55/5 20/5  Dysplasia (Low/High)

Stool Collection

Stools were collected more than 2 weeks following any colorectaldiagnostic procedure or cathartic preparation and prior to eitherendoscopic or surgical neoplasm resection. Patients collected wholestools in a preservative buffer (0.5 mol/L Tris, 10 mmol/L NaCl, 150mmol/L EDTA, pH 9.0) as described (see, e.g., Olson J, et al., Diagn MolPathol 2005; 14:183-191; herein incorporated by reference in itsentirety), and mailed to a laboratory within 48 hours. Once a stoolarrived in the laboratory, it was weighed and homogenized. One aliquotequivalent to 10 g stool was used for stool DNA extraction, and the restwas stored at −80° C. in aliquots.

Long DNA Quantification with Real-Time Alu PCR

Crude stool DNA was extracted with isopropanol, precipitated withethanol, and eluted in 7.5 ml 1×TE buffer. Human DNA in crude stool DNAwas quantified using a real-time Alu PCR method (see, e.g., Zou H, etal., Cancer Epidemiol Biomarkers Prev 2006; 15:1115-1119; hereinincorporated by reference in its entirety). Primers specific for thehuman Alu sequences, sense: 5′-ACG CCT GTA ATC CCA GCA CTT-3; andantisense: 5′-TCG CCC AGG CTG GAG TGC A-3′ were used to amplifysequences about 245 bp inside Alu repeats (see, e.g., Zou H, et al.,Cancer Epidemiol Biomarkers Prev 2006; 15:1115-1119; Zijlstra A, et al.,Cancer Res 2002; 62:7083-7092; each herein incorporated by reference intheir entireties). Crude stool DNA was diluted 1 to 100 withnuclease-free water for PCR amplification. One μL water-diluted stoolDNA was amplified in a total volume of 25 μL containing 1×iQ™ SYBR®Green Supermix (BioRad), 200 nM each primer under the followingconditions: 95° C. for 3 minutes, followed by 30 cycles of 95° C. for 30seconds, 60° C. for 30 seconds, and 72° C. for 40 seconds in a real-timeiCycler® (BioRad). Standard curve was created for each plate byamplifying 10-fold serially diluted human genomic DNA samples (Novagen,Madison, Wis.). Melting curve was made after each PCR reaction toconfirm that only one product was amplified for all samples.Amplification was carried out in 96-well plates. Each plate consisted ofstool DNA samples and multiple positive and negative controls. Eachassay was performed in duplicate.

Fecal Occult Blood Quantification with HemoQuant

Fecal occult blood was quantified with HemoQuant test (see, e.g.,Harewood G C, et al., Mayo Clin Proc 2002; 77:23-28; Ahlquist D A, etal., JAMA 1993; 269:1262-1267; Ahlquist D A, et al., N Engl J Med 1985;312:1422-1428; Ahlquist D A, et al., Ann Intern Med 1984; 101:297-302;Schwartz S, et al., Clin Chem 1983; 29:2061-2067; Schwartz S, et al.,Gastroenterology 1985; 89:19-26; each herein incorporated by referencein their entireties). Buffer (0.5 mol/L Tris, 10 mmol/L NaCl, 150 mmol/LEDTA, pH 9.0) (see, e.g., Olson J, et al., Diagn Mol Pathol 2005;14:183-191; herein incorporated by reference in its entirety) preservedfrozen stool slurry equivalent to 8 mg stool was used to performHemoQuant test with an automated system in a clinical lab.Non-fluorescing heme in stool sample was converted to fluorescingporphyrins by removal of iron with 2 mL hot (60° C.) oxalic acid and 0.2mL ferrous sulfate. The reaction mixture was then extracted with 3 mLethyl acetate:isobutyl alcohol (11.1:1, v/v) followed by washing with 3mL alkalinized aqueous solution of potassium acetate, and furtherextracted with 2.5 mL acetic acid/phosphoric acid. The extractedporphyrins, mostly protoporphyrin, are quantified based on fluorescenceemission intensity at 652 nm using 405 nm as the excitation wavelengthin an Infinite® 200 microplate reader (Tecan, Männedorf, Switzerland).Each plate consisted stool samples, standards, and negative and positivecontrols. Standards were made with known amounts of hemoglobin fromhuman blood.

Statistical Analysis

Logistic procedure was used to calculate the correlation of stool longDNA and hemoglobin levels. Wilcoxon Rank Sum test was used to comparethe long DNA or hemoglobin levels between each of the three differentstool groups, and evaluate the association of marker levels with tumorlocation, gender, Dukes stage, and differentiation grade. Thecorrelation of marker levels with tumor size and patient age wascalculated with Logistic procedure. Chi-Square and Fisher exact testswere used to evaluate the association of detection rates of marker panelwith clinical characteristics. Combination of long DNA and hemoglobinlevels was calculated with a logistic model. Receiver Operating Curve(ROC) was constructed to compare long DNA and hemoglobin levels incancers or adenomas versus normal subjects, and area under the curve(AUC) value was also calculated for each curve. Sensitivities werecalculated at 90% specificity for single markers and their combination.Statistical analysis was conducted with SAS software (SAS Institute,Cary, N.C.).

Results

The median human DNA levels were 421 (range; 0-19140), 86 (0-6260), and11 (0-30000) ng per g stools from patients with CRCs or adenomas, orfrom normal controls, respectively (p=0.0001, CRC vs normal; p=0.0005,adenoma vs. normal; p=0.007, CRC vs adenoma; FIG. 1A). The medianhemoglobin levels were 3.4 (0.1-36.1), 1.5 (0.1-10.9), and 1.1(0.3-11.0) mg per g stools from patients with CRCs or adenomas, or fromnormal controls, respectively (p=0.0001, CRC vs. normal or adenoma;p=1.0, adenoma vs normal; FIG. 1B). Fecal long DNA and occult bloodlevels were not correlated (R²=0.0001; FIG. 2).

At 90% specificities, long DNA testing detected 70% of colorectalcancers and 46% of adenomas while occult blood testing detected 50% and12% (FIGS. 1 & 3). Combining these tests detected 80% colorectal cancersand 46% of adenomas at 90% specificity (FIG. 3). For detection ofcolorectal cancer, AUC values were 0.82, 0.78, and 0.90 for fecal longDNA, occult blood, and combination testing, respectively (p=0.02, longDNA vs combination; p=0.0001, occult blood vs. combination; FIG. 3). Fordetection of advanced adenoma, AUC values were 0.72, 0.50, and 0.72 forfecal DNA, occult blood, and combination testing, respectively (p=0.8,long DNA vs combination; p=0.03, occult blood vs. combination; FIG. 3).

The median (range) fecal long DNA level was 181 (0-7120) ng/g stool withstage 1-2 cancers and 910 (0-19140) ng/g stool with stage 3-4 cancers,p=0.001; and 112 (0-4780) ng/g stool with proximal cancers and 1006(0-19140) ng/g stool with distal cancers, p=0.0001. The median fecaloccult blood level was 7.0 mg Hb/g (0.2-26.4 mg/g) with proximal cancersand 2.5 mg/g (0.1-36.1 mg/g) with distal ones, p=0.04. The median fecallong DNA and occult blood levels in stools from patients with CRCs andadvanced adenomas were not associated with other clinicalcharacteristics. Median size was 4.0 cm (1.0-15.0) for neoplasmsdetected by the combined tests and 3.7 cm (1.0-7.0) for neoplasmsmissed, p=0.02. Neoplasm detection rates by combined tests were affectedby neither tumor site nor stage.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

We claim:
 1. A method for detecting the presence of a colorectalneoplasm in a mammal, said method comprising: a) obtaining a stoolsample from said mammal; b) detecting the presence or absence of one ormore exfoliated epithelial markers in said stool sample, wherein thepresence of one or more exfoliated epithelial markers in said stoolsample is indicative of a colorectal neoplasm in said mammal; and c)detecting the presence or absence of one or more fecal occult bloodmarkers in said stool sample, wherein the presence of said one or morefecal occult blood markers specific for a colorectal neoplasm in saidstool sample is indicative of a colorectal neoplasm in said mammal.